Protein citrullination (deimination), a post-translational modification (PTM) altering the amino acid residue arginine (Arg), has recently become an area of interest due its emerging role in human auto-immune diseases. We are the first group to establish that this PTM is present in the human heart. Specifically, our preliminary data shows citrullination occurs in myofilament proteins such as tropomyosin (Tm), myosin (heavy and light chain) and myosin binding protein C. Furthermore, we find an increase in the extent of deiminated proteins in myocardium obtained from patients with heart failure (HF) compared to control hearts. Based on these results, we hypothesize that citrullination plays a role in the pathogenesis of HF, particularly affecting myocardial contraction. Furthermore, we hypothesize that citrullination is exacerbated in HF especially when there is an autoimmune component. This exploratory grant is aimed to establish the relation between protein citrullination, peptidyl arginine deiminase (PAD), the enzyme responsible for this PTM, and cardiac and myocardial dysfunction in the failing heart. In Specific Aim 1, PAD isoform expression and citrullinated myocardial proteins are explored in three mouse models of HF, chronic aortic constriction and two inflammatory dilated cardiomyopathies. Using a recently developed mass spectrometry-based method, citrullinated proteins and the modified amino acid residues will be identified and quantified. In addition, using a combination of molecular biology and a second innovative mass spectroscopy approach, we will unambiguously define and quantify the PAD isoforms in cardiac myocytes. Specific aim 2 focuses on the assessment of the functional role of PAD and protein citrullination in cardiac myocytes. Combing molecular biology and pharmacological methods to manipulate PAD isoform activity, the extent of modified proteins is correlated to myocardial contractile function. Tm mutants with Ala (non modifiable) or Gln (a potential mimetic of Arg-Cit) replacing the modifiable citrullinatable Arg residues, will be tested for functional alterations contraction. This program is highly novel as protein citrullination and PAD system has never been explored in the heart, yet our new evidence supports its potential to modulate muscle contractility. This work will lead to understanding of an entirely new mechanism for post-translational protein regulation in the heart. PUBLIC HEALTH RELEVANCE: For the first time, we show that protein citrullination (deimination), a post-translational modification (altering the amino acid residue arginine (Arg) occurs in the heart, targeting a number of contractile proteins, and that it increases with heart failure. This exploratory R21 proposal aims to establish the relation between protein citrullination, peptidyl arginine deiminase (PAD), the enzyme responsible for this PTM, and myocardial contractile dysfunction in three different models of HF. This highly novel program will lead to understanding of an entirely new mechanism for post-translational protein regulation in the heart.